Ki-67 protein concentrations in urothelial bladder carcinomas are related to Ki-67-specific RNA Concentrations in Urine.
Spontaneously voided clean-catch urines from 68 patients were collected with informed consent, stored at 4[degrees]C, and processed within 4 h of collection. Approval was obtained from the Ethikkommission of the University of Luebeck. Three groups of patients were included: (a) healthy donors (n = 14); (b) patients with urinary tract infection as detected by analysis of urine sediment (n = 28); and (c) patients with bladder carcinoma (n = 26). All urine samples from patients with tumors were checked for significant bacteriuria or leukocyturia (>10/[micro]L) as well as for the presence of erythrocytes (>10/[micro]L). The specific weights of all urine samples were in the range 1.010-1.015 kg/L, showing that there were no major differences in urine concentration. After routine diagnostics were completed, 1 mL of urine was mixed 1:1 with lysis buffer [5.64 mol/L guanidinium thiocyanate, 5 g/L sarcosyl, 50 mmol/L sodium acetate (pH 6.5), 1 mmol/L [beta]-mercaptoethanol], the pH was adjusted to 7 by addition of 1.5 mol/L HEPES (pH 8.0), and samples were frozen at -80[degrees]C until RNA extraction was performed.
RNA was isolated by use of the RNeasy Midi reagent set (Qiagen), according to the manufacturer's instructions except for the fact that lysis buffer (see above) was used instead of buffer RLT. We subjected 200 [micro]L of the product from the RNeasy extraction procedure to DNase I digestion, inactivated the DNase by incubation for 10 min at 65[degrees]C in the presence of 2.5 mmol/L EDTA, precipitated the RNA in ethanol, and resuspended the pellet in 7 [micro]L of deionized water. The complete isolate was reverse-transcribed with use of random hexamer priming and superscript 11 reverse transcriptase (Invitrogen) according to the manufacturer's protocol at 42[degrees]C in a total volume of 20 [micro]L. The enzyme was inactivated for 15 min at 72[degrees]C, and the cDNA was stored at -80[degrees]C until use.
Primers and a TagMan probe for the quantitative real-time PCR were constructed with Primer Express, Ver. 2.0 (Applied Biosystems), with the Ki-67 mRNA sequence from the National Center for Biotechnology Information database (NM 002417). The sequences were as follows: Oligo 1 (19mer), 5'-CGG ACT TTG GGT GCG ACT T-3'; Oligo 2 (18mer), 5'-GTC GAC CCC GCT CCT TTT-3'; and TagMan Oligo, 5'-6FAM-ACG AGC GGT GGT TCG ACA AGT GTXTP-3', where FAM is 6-carboxyfluorescein and X is 6-carboxytetramethyl rhodamine.
Each quantitative PCR assay contained 2.5 [micro]L of 10x buffer from Eurogentec, 5 mM Mg[Cl.sub.2], 200 [micro]M deoxynucleotide triphosphate mixture, 300 nM each primer, 100 nM TagMan Probe, 0.125 [micro]L of HotGoldStar enzyme (Eurogentec), and 2 /,L of cDNA in a total volume of 25 [micro]L. The cDNA solution was diluted threefold before use in the PCR reaction. To take into account the dilution steps, we multiplied the quantitative PCR values by a factor of 30 to give the copy number/mL of urine. In addition, we added 0.5 pg of carrier DNA to all reactions, including nontemplate controls, to avoid nonspecific adsorption of cDNA to the plastic ware. The two-step PCR reactions included 50 cycles of annealing and denaturation at 60[degrees]C and 95[degrees]C, respectively. Reactions were carried out on a Gene Amp 5700[TM] (Applied Biosystems). To be able to transform the threshold cycle values into absolute RNA copy numbers, we constructed a calibration curve from a dilution series of a gel-purified restriction fragment of Ki-67 DNA (see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue8/). PCR reactions were performed in duplicate for each cDNA, and mean values were used for further calculations if the values did not differ more than 50%. In the case of a larger variation, we performed a second independent PCR reaction, and the mean value of all four reactions was used. As we have described previously (5), the overall assay variation (based on three independent experiments performed for different urines) was less than a factor of 2. Two copies per PCR tube were readily detected, giving a mean (SD) threshold cycle value of 38.9 (0.8) cycles (see Fig. 1 in the online Data Supplement). The nontemplate controls showed no threshold cycle values <50, indicating only a small amount of nonspecific amplification (data not shown). All tumors were graded according to the WHO classification (6) and staged according to the criteria formulated by the International Union against Cancer (7). Shown in Fig. 1A is a box plot of the Ki-67 RNA content in urine, according to patient group: healthy controls, patients with urinary tract infection, and patients with bladder carcinoma (stratified according to tumor grade).
[FIGURE 1 OMITTED]
The amounts of Ki-67 RNA in urine from patients with grade 2 and 3 carcinomas were significantly higher than those in urine from healthy controls. Moreover, they differed significantly from the amounts of Ki-67 RNA in urine from patients with urinary tract infections or patients with grade 1 carcinomas. Among the 14 healthy individuals tested, there were only 3 cases of detectable Ki-67 RNA sequences. We observed no statistically significant difference between Ki-67 RNA concentrations in urine from patients with grade 3 carcinomas and patients with grade 2 carcinomas, as indicated by a P value of 0.63. For all samples from tumor patients, significant bacteriuria or leukocyturia as well as the presence of erythrocytes could be excluded except for two G1 tumor samples. In these samples, the Ki-67 RNA concentrations in urine were 16 and 68 copies/mL, which are near the median (32 copies/mL) of the group with grade G1 cancer. We did not find a statistically significant difference between the amount of Ki-67 RNA in urine from patients with grade 1 carcinomas compared with the controls with urinary tract infections (P = 0.092).
For the 26 patients with bladder carcinoma, tumor tissue was obtained by transurethral resection or cystectomy and fixed in 45 mL/L buffered formaldehyde for histopathologic analysis. Ki-67 indices of tumor samples were determined on paraffin sections stained immunohistochemically with the MIB-1 antibody as described previously (8). In each case, the number of Ki-67-positive cells (indicated by brown nuclear staining) among 200 tumor cells was counted. The Ki-67 index was calculated as the proportion of Ki-67-positive cells to the total number of cells counted. Because proliferative activity varied within different areas in some tumors, only those areas with the highest activity were analyzed. Paraffin blocks from four cases were not available for immunohistochemical analysis; therefore, Ki-67 labeling indices could be determined for only 22 of 26 bladder cancer patients. As demonstrated previously (2-4), the labeling index is correlated to the grade of the carcinoma, being highest in grade 3 tumor material (data not shown). We next sought to examine the relationship between the Ki-67 RNA copy number in urine and the Ki-67 protein labeling index of the corresponding tumor material. This relationship for the individual samples is illustrated in Fig. 1B. The two indicators were significantly correlated (P = 0.009; r = 0.55) as determined by the Spearmen p rank correlation test.
Finally, we evaluated the diagnostic significance of the individual Ki-67 copy numbers determined in the urine of tumor patients by performing ROC curve analysis. For the detection of a bladder carcinoma (regardless of tumor grade), the area under the ROC curve was calculated to be 0.71 (95% confidence interval, 0.59-0.84). When grade G2 and G3 carcinomas were grouped together, the area under the ROC curve was 0.83 (95% confidence interval, 0.71-0.95). At a cutoff value of 90 copies/mL of urine, among the 17 samples with grade G2 and G3 carcinomas, 13 of 17 samples (76%) were detected correctly, whereas 41 of the 52 control samples (79%) were correctly classified as negative.
In this report we describe for the first time the correlation between an established protein marker in tumor tissue and the corresponding RNA content in total urine. In contrast to other studies [e.g., Refs. (9-13)], we decided to take total urine as the source for RNA isolation to include the possibility that there might be sources other than shed cells contributing to the isolated RNA. Although a larger number of samples must be studied, the demonstrated correlation between the Ki-67 labeling index in tumor material and the Ki-67 RNA copy number in total urine offers the prospect of transferring the prognostic potential of the Ki-67 labeling index to the noninvasive diagnostic tool of RNA quantification in urine.
We thank Drs. T. Dann, 5. Thomas, M. Horn, and J-M. Traeder for collecting samples; W. Wuensche for technical assistance; and C.C. Prawda for helpful discussions and critical reading of the manuscript.
(1.) Scholzen T, Endl E, Wohlenberg C, van der Sar S, Cowell IG, Gerdes J, et al. The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure. J Pathol 2002;196:135-44.
(2.) Oosterhuis JW, Schapers RF, Janssen-Heijnen ML, Smeets AW, Pauwels RP. MIB-1 as a proliferative marker in transitional cell carcinoma of the bladder: clinical significance and comparison with other prognostic factors. Cancer 2000;88:2598-605.
(3.) Suwa Y, Takano Y, IN M, Asakura T, Noguchi S, Masuda M. Prognostic significance of Ki-67 expression in transitional cell bladder carcinoma after radical cystectomy. Pathol Res Pract 1997;193:551-6.
(4.) Santos L, Amaro T, Costa C, Pereira S, Bento MJ, Lopes P, et al. Ki-67 index enhances the prognostic accuracy of the urothelial superficial bladder carcinoma risk group classification. Int J Cancer 2003;105:267-72.
(5.) Menke TB, Warnecke JM. Improved conditions for isolation and quantification of RNA in urine specimen. Ann N Y Acad Sci; in press.
(6.) Mostofi FK, Davis CJ Jr, Sesterhenn IA. Histological typing of urinary bladder tumors. World Health Organization--international histological classification of tumors. Berlin: Springer, 1999:10-1.
(7.) Sobin LH, Wittekind C, eds. TNM classification of malignant tumors, 6th ed. Berlin: Springer, 2002:187-9.
(8.) Knuger S, Muller H. Correlation of morphometry, nucleolar organizer regions, proliferating cell nuclear antigen and Ki-67 antigen expression with grading and staging in urinary bladder carcinomas. Br J Urol 1995;75:480-4.
(9.) Bialkowska-Hobrzanska H, Bowles L, Bukala B, Joseph MG, Fletcher R, Razvi H. Comparison of human telomerase reverse transcriptase messenger RNA and telomerase activity as urine markers for diagnosis of bladder carcinoma. Mol Diagn 2000;4:267-77.
(10.) Miyake H, Etc H, Arakawa S, Kamidono S, Hara I. Over expression of CD44V8-10 in urinary exfoliated cells as an independent prognostic predictor in patients with urothelial cancer. J Urol 2002;167:1282-7.
(11.) Okegawa T, Kinjo M, Horie S, Nutahara K, Higashihara E. Detection of mucin 7 gene expression in exfoliated cells in urine from patients with bladder tumor. Urology 2003;2:182-6.
(12.) Chiu AW, Huang YL, Huan SK, Wang YC, Ju JP, Chen MF, et al. Potential molecular marker for detecting transitional cell carcinoma. Urology 2002; 60:181-5.
(13.) Hotakainen K, Haglund C, Paju A, Nordling S, Alfthan H, Rintala E, et al. Chorionic gonadotropin R-subunit and core fragment in bladder cancer: mRNA and protein expression in urine, serum and tissue. Eur Urol 2002; 41:677-85.
Tim B. Menke,  Katrin Boettcher,  Stefan Kriiger,  Ingo Kausch,  Andreas Boehle,  Georg Sczakiel,  and Jens M. Warnecke  * ( Institut fuer Molekulare Medizin,  Institut fuer Pathologie, and  Klinik and Poliklinik fuer Urologie, UK-SH, Campus Luebeck and Universitaet zu Luebeck, Luebeck, Germany;  HELIOS Agnes Karll Krankenhaus, Bad Schwartau, Germany; * address correspondence to this author at: Institut fuer Molekulare Medizin, Universitaet zu Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany; fax 49-451-500-2729, e-mail email@example.com. de)
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|Title Annotation:||Technical Briefs|
|Author:||Menke, Tim B.; Boettcher, Katrin; Kruger, Stefan; Kausch, Ingo; Boehle, Andreas; Sczakiel, Georg; Wa|
|Date:||Aug 1, 2004|
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